The instant invention concerns novel chromophore containing compounds useful in determining Interleukin-1xcex2 convertase activity. Interleukin-1xcex2 convertase (ICE) has been identified as the enzyme responsible for converting precursor interleukin-1xcex2 (IL-1xcex2) to biologically active IL-1xcex2. Compounds of Formulas I and II are useful in the diagnosing and monitoring IL-1 mediated dieases or in evaluation inhibitors of interleukin-1xcex2 convertase.
Mammalian interleukin-1 (IL-1) is an immunoregulatory protein secreted by cell types as part of the inflammatory response. The primary cell type responsible for IL-1 production is the peripheral blood monocyte. Other cell types have also been described as releasing or containing IL-1 or IL-1 like molecules. These include epithelial cells (Luger, et al., J. Immunol. 127: 1493-1498 (1981), Le et al., J. Immunol. 138: 2520-2526 (1987) and Lovett and Larsen, J. Clin. Invest. 82: 115-122 (1988), connective tissue cells (Ollivierre et al., Biochem. Biophys. Res. Comm. 141: 904-911 (1986), Le et al, J. Immunol. 138: 2520-2526 (1987), cells of neuronal origin (Giulian et al., J. Esp. Med. 164: 594-604 (1986) and leukocytes (Pistoia et al., J. Immunol. 136: 1688-1692 (1986), Acres et al., Mol. Immuno. 24: 479-485 (1987), Acres et al., J. Immunol. 138: 2132-2136 (1987) and Lindenmann et al., J. Immunol 140: 837-839 (1988).
Biologically active IL-1 exists in two distinct forms, IL-1xcex1 with an isoelectric point of about pI 5.2 and IL-1xcex2 with an isoelectric point of about 7.0 with both forms having a molecular mass of about 17,500 (Bayne et al., J. Esp. Med. 163: 1267-1280 (1986) and Schmidt, J. Esp. Med. 160: 772 (1984). The polypeptides appear evolutionarily conserved, showing about 27-33% homology at the amino acid level (Clark et al., Nucleic Acids Res. 14: 7897-7914 (1986).
Mammalian IL-1 is synthesized as a cell associated precursor polypeptide with a molecular mass of about 31.4 kDa (Limjuco et al., Proc. Natl. Acad. Sci USA 83: 3972-3976 (1986). Precursor IL-1xcex2 is unable to bind to IL-1 receptors and is biologically inactive (Mosley et al., J. Biol. Chem. 262: 2941-2944 (1987). Biological activity appears dependent upon some form of proteolytic processing which results in the conversion of the precursor 31.5 kDa form to the mature 17.5 kDa form. Evidence is growing that by inhibiting the conversion of precursor IL-1xcex2 to mature IL-1xcex2, one can effectively inhibit the activity of interleukin-1.
Mammalian cells capable of producing IL-1xcex2 include, but are not limited to, karatinocytes, endothelial cells, mesangial cells, thymic epithelial cells, dermal fibroblasts, chondrocytes, astrocytes, glioma cells, mononuclear phagocytes, granulocytes, T and B lymphocytes and NK cells.
As discussed by J. J. Oppenheim, et al. Immunology Today, vol. 7(2):45-56 (1986), the activities of interleukin-l are many. It has been observed that catabolin, a factor that promotes degradation of cartilage matrix, also exhibited the thymocyte comitogenic activities Qf IL-1 and stimulates chondrocytes to release collagenase neutral proteases and plasminogen activator. In addition, a plasma factor termed proteolysis inducing factor stimulates muscle cells to produce prostaglandins which in turn leads to proteolysis, the release of amino acids and, in the long run, muscle wasting, and appears to represent a fragment of IL-1 with fever-inducing, acute phase response and thymocyte co-mitogenic activities.
IL-1 has multiple effects on cells involved in inflammation and wound healing. Subcutaneous injection of IL-1 leads to margination of neutrophils and maximal extravascular infiltration of the polymorphonuclear leukocytes (PMN). In vitro studies reveal IL-1 to be a chemotactic attractant for PMN to activate PMN to metabolize glucose more rapidly to reduce nitroblue tetrazolium and to release their lysozomal enzymes. Endothelial cells are stimulated to proliferate by IL-1 to produce thromboxane, to become more adhesive and to release procoagulant activity. IL-1 also enhances collagen type IV production by epidermal cells, induces osteoblast proliferation and alkaline phosphatase production and stimulates osteoclasts to resorb bone. Even macrophages have been reported to be chemotactically attracted to IL-1 to produce prostaglandins in response to IL-1 and to exhibit a more prolonged and active tumoricidal state.
IL-1xcex2 is also a potent bone resorptive agent capable upon infusion into mice of causing hypercaleemia and increas in bone resorptive surface as revealed by his to morphometry Sabatini, M. et al., PNAS 85: 5235-5239, 1988.
Accordingly, IL-1 has been implicated in infectious diseases where active infection exists at any body site, such as meningitis and salpingitis; complications of infections including septic shock, disseminated intravascular coagulation, and/or adult respiratory distress syndrome; acute or chronic inflammation due to antigen, antibody, and/or complement deposition; inflammatory conditions including arthritis, cholangitis, colitis, encephalitis, endocarditis, glomerulonephritis, hepatitis, myocarditis, pancreatitis, pericarditis, reperfusion injury and vasculitis. Immune-based diseases which may be responsive to ICE inhibitors of Formula I include but are not limited to conditions involving T-cells and/or macrophages such as acute and delayed hypersensitivity, graft rejection, and graft-versus-host-disease; auto-immune diseases including Type I diabetes mellitus and multiple sclerosis. IL-1 has also been implicated in the treatment of bone and cartilage resorption as well as diseases resulting in excessive deposition of extracellular matrix. Such diseases include periodonate diseases interstitial pulmonary fibrosis, cirrhosis, systemic sclerosis, and keloid formation.
Disclosed are novel chromophore containing compounds of Formula I and their use in determining interleukin-1xcex2 convertase (ICE) activity. Compounds of Formula II are useful in the diagnosing and monitoring IL-1 mediated dieases or in evaluation inhibitors of interleukin-1xcex2 convertase. 
In one embodiment the invention concerns a chromophore compound of Formula I 
wherein:
AA1, is independently selected from the group consisting of
(a) a single bond, and
(b) an amino acid of formula AI 
xe2x80x83wherein R1 selected from the group consisting of
(a) hydrogen,
(b) substituted C1-6 alkyl, wherein the substituent is selected from
(1) hydrogen,
(2) hydroxy,
(3) halo,
(4) xe2x80x94Sxe2x80x94C1-4 alkyl,
(5) xe2x80x94SH
(6) C1-6 alkylcarbonyl,
(7) carboxy, 
(9) C1-4 alkylamino, wherein the alkyl moeity is substituted with hydrogen or hydroxy, and the amino is substituted with hydrogen or CBZ,
(10) guanidino,
(11) amino, and
(c) aryl C1-6 alkyl or substituted aryl C1-6 alkyl wherein,
the aryl group is selected from the group consisting of:
(a) phenyl,
(b) naphthyl,
(c) pyridyl,
(d) furyl,
(e) thienyl,
(f) thiazolyl,
(g) isothiazolyl,
(h) imidazolyl,
(i) benzimidazolyl,
(j) pyrazinyl,
(k) pyrimidyl,
(l) quinolyl,
(m) isoquinolyl,
(n) benzofuryl,
(o) benzothienyl,
(p) pyrazolyl,
(q) indolyl,
(r) purinyl,
(s) isoxazolyl, and
(t) oxazolyl, and mono and di-substituted aryl as defined above in items (a) to (t) wherein the substitutents are independently C1-6alkyl, halo, hydroxy, C1-6alkyl amino, C1-6alkoxy, C1-6alkylthio, and C1-6alkylcarbonyl;
AA2 is independently selected from the group consisting of
(a) a single bond, and
(b) an amino acid of formula AII 
AA3, which are each independently selected from the group consisting of
(a) a single bond, and
(b) an amino acid of formula AIII 
xe2x80x83wherein R2 and R3 are each independently selected from the group consisting of
(a) hydrogen,
(b) substituted C1-6alkyl, wherein the substituent is selected from
(1) hydrogen,
(2) hydroxy,
(3) halo,
(4) xe2x80x94Sxe2x80x94C1-4alkyl,
(5) xe2x80x94SH
(6) C1-6 alkylcarbonyl,
(7) carboxy, 
(9) C1-4 alkylamino, wherein the alkyl moeity is substituted with hydrogen or hydroxy, and the amino is substituted with hydrogen or CBZ,
(10) guanidino,
(11) amino, and
(c) aryl C1-6 alkyl,
wherein aryl is defined as immediately above, and wherein the aryl may be mono and di-substituted, the substituents being each independently C1-6alkyl, halo, hydroxy, C1-6alkyl amino, C1-6alkoxy, C1-6alkylthio, and C1-6alkylcarbonyl;
n is an interger from 0-16 and
(AA)n is a peptide of 0-16 (ie n) amino acids in length, each amino acid being independent of formula 
xe2x80x83wherein X selected from the group consisting of
(a) hydrogen,
(b) substituted C1-6 alkyl, wherein the substituent is selected from
(1) hydrogen,
(2) hydroxy,
(3) halo,
(4) xe2x80x94Sxe2x80x94C1-4 alkyl,
(5) xe2x80x94SH
(6) C1-6 alkylcarbonyl,
(7) carboxy, 
(9) amino carbonyl amino,
(10) C1-4 alkylamino, wherein the alkyl moeity is substituted with hydrogen or hydroxy, and the amino is substituted with hydrogen or CBZ,
(11) guanidino,
(12) amino, and
(c) aryl C1-6alkyl,
wherein the aryl group is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl,
(4) furyl,
(5) thienyl,
(6) thiazolyl,
(7) isothiazolyl,
(8) imidazolyl,
(9) benzimidazolyl,
(10) pyrazinyl,
(11) pyrimidyl,
(12) quinolyl,
(13) isoquinolyl,
(14) benzofuryl,
(15) benzothienyl,
(16) pyrazolyl,
(17) indolyl,
(18) purinyl,
(19) isoxazolyl, and
(20) oxazolyl,
and wherein the aryl may be mono and di-substituted, the substituents being each independently C1-6alkyl, halo, hydroxy, C1-6alkyl amino, C1-6alkoxy, C1-6alkylthio, and C1-6alkylcarbonyl;
R5 is
(a) substituted C1-12 alkyl, wherein the substituent is selected from
(1) hydrogen,
(2) hydroxy,
(3) halo, and
(4) C1-6 alkylcarbonyl;
(b) aryl C1-6 alkyl wherein the aryl group is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl,
(4) furyl,
(5) thienyl,
(6) thiazolyl,
(7) isothiazolyl,
(8) imidazolyl,
(9) benzimidazolyl,
(10) pyrazinyl,
(11) pyrimidyl,
(12) quinolyl,
(13) isoquinolyl,
(14) benzofuryl,
(15) benzothienyl,
(16) pyrazolyl,
(17) indolyl,
(18) purinyl,
(19) isoxazolyl, and
(20) oxazolyl,
and mono and di-substituted aryl as defined above in items (1) to (20) wherein the substitutents are independently C1-6alkyl, halo, hydroxy, C1-6alkyl amino, C1-6alkoxy, C1-6alkylthio, and C1-6alkylcarbonyl.
R6 is selected from the group consisting of:
(a) mono, di or tri substituted Aryl amino,
(b) mono, di or tri substituted Aryl oxy, and
(c) mono, di or tri substituted Aryl thio,
wherein the aryl group is selected from the group consisting of: 
wherein the substituent is selected from the group consisting of
(1) H,
(2) OH,
(3) halo,
(4) C1-6alkyl,
(5) C1-6alkyloxy,
(6) CO2H,
(7) NO2 
(8) SO3H
(9) formyl,
(10) NH2,
(11) SH,
(12) C1-6alkylthio, 
(14) phenyl,
(15) phenylC1-6alkyl
(16) NO,
(17) C1-6alkylcarbonyl,
(18) phenylazo,
(19) C1-6sulfinyl,
(20) C1-6sulphonyl,
(21) phenyl sulfinyl,
(22) phenyl sulfonyl,
(23) phenyl carbonyl,
(24) phenyl oxy,
(25) phenyl thiol,
(26) C1-4alkylamino,
(27) diC1-4alkylamino, and
(28) CN,
and R8 is
C1-6alkyl or aryl C1-6alkyl wherein aryl is selected from the phenyl and naphthyl.
As above, (AA)n defines a peptide of 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15, or 16 amino acids in length. Similarly, for purposes of this specification, the amino acids AAI, AAII, and AAIII may be each independently selected from the group consisting of the L- and D- forms of the amino acids including glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxy-lysine, histidine, arginine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, ornithine, xcex2-alanine, homoserine, homotyrosine, homophenylalanine and citrulline. Compounds of Formula I correspond to the peptide sequence which is SEQ. ID NO: 1:
In one class of the first embodiment n is 0. Within this class is the subclass
wherein
AA1, is an amino acid of formula 
AA2 is an amino acid of formula 
AA3 is an amino acid of formula 
Within the subclass are the compounds wherein R2 and R3 are each independently selected from the group consisting of
(a) hydrogen,
(b) C1-6alkyl, wherein the substituent is selected from
(1) hydrogen,
(2) hydroxy,
(3) halo,
(4) xe2x80x94Sxe2x80x94C1-4alkyl,
(5) xe2x80x94SH,
(6) C1-6 alkylcarbonyl,
(7) carboxy, 
(9) C1-4alkylamino, and C1-4 alkyl amino wherein the alkyl moeity is substituted with an hydroxy, and
(10) guanidino,
(11) amino, and
(c) aryl C1-6 alkyl,
wherein aryl is phenyl, naphthyl, pyridyl, furyl, thienyl, thiazolyl, isothiazolyl, benzofuryl, benzothienyl, indolyl, isoxazolyl, and oxazolyl; and wherein the aryl may be mono and di-substituted, the substituents being each independently C1-6alkyl, halo, hydroxy, C1-6alkyl amino, C1-6alkoxy, C1-6alkylthio, and C1-6alkylcarbonyl.
R6 is selected from the group consisting of:
(a) mono or di substituted Aryl amino,
(b) mono or di substituted Aryl oxy, and
(c) mono or di substituted Aryl thio,
wherein the aryl group is selected from the group consisting of: 
wherein the substituent is selected from the group consisting of
(1) H,
(2) OH,
(3) halo,
(4) C1-6alkyl,
(5) C1-6alkyloxy,
(6) CO2H,
(7) NO2,
(8) SO3H,
(9) formyl, and
(10) CN.
More particularly, illustrating the invention are the compounds wherein: R5 is methyl;
R2 is C1-6alkyl; and
R3 is
(a) hydrogen,
(b) C1-6alkyl,
(d) benzyl,
(e) p-hydroxy-benzyl,
(f) N-carbobenzoxy-amino-(n-butyl),
(g) carbamylmethyl,
(h) carbamylethyl,
(i) indol-2-yl-methyl,
(j) substituted phenyl C1-6alkyl, wherein the substituent is hydrogen, hydroxy, carboxy, or C1-4alkyl,
(k) substituted indolyl C1-6alkyl, wherein the substituent is hydrogen, hydroxy, carboxy, or C1-4alkyl, or
(1) substituted imidazolyl C1-6alkyl wherein the substituent is hydrogen, hydroxy, carboxy, or C1-4alkyl.
In a second embodiment the invention concerns a chromophore containing compound of Formula II 
wherein:
AA1, is independently selected from the group consisting of
(a) a single bond, and
(b) an amino acid of formula AI 
xe2x80x83wherein R1 selected from the group consisting of
(1) substituted C1-6 alkyl, wherein the substituent is selected from
(a) hydrogen,
(b) hydroxy,
(c) halo,
(d) C1-6alkyl carbonyl, and
(e) amino,
(2) aryl C1-6 alkyl or substituted aryl C1-6alkyl wherein, the aryl group is selected from the group consisting of:
(a) phenyl,
(b) naphthyl,
(c) pyridyl,
(d) furyl,
(e) thienyl,
(f) thiazolyl,
(g) isothiazolyl,
(h) imidazolyl,
(i) benzimidazolyl,
(j) pyrazinyl,
(k) pyrimidyl,
(l) quinolyl,
(m) isoquinolyl,
(n) benzofuryl,
(o) benzothienyl,
(p) pyrazolyl,
(q) indolyl,
(r) purinyl,
(s) isoxazolyl, and
(t) oxazolyl,
and mono and di-substituted aryl as defined above in items (a) to (t) wherein the substitutents are independently C1-6alkyl, halo, hydroxy, C1-6alkyl amino, C1-6alkoxy, C1-6alkylthio, and C1-6alkylcarbonyl;
AA2 is independently selected from the group consisting of
(a) a single bond, and
(b) an amino acid of formula AII 
AA3, which are each independently selected from the group consisting of
(a) a single bond, and
(b) an amino acid of formula AIII 
xe2x80x83wherein R2 and R3 are each independently selected from the group consisting of
(a) hydrogen,
(b) substituted C1-6alkyl, wherein the substituent is selected from
(1) hydrogen,
(2) hydroxy,
(3) halo,
(4) xe2x80x94Sxe2x80x94C1-4alkyl,
(5) xe2x80x94SH
(6) C1-6 alkylcarbonyl,
(7) carboxy, 
(9) C1-4 alkylamino, wherein the alkyl moeity is substituted with hydrogen or hydroxy, and the amino is substituted with hydrogen or CBZ,
(10) guanidino,
(11) amino, and
(c) aryl C1-6 alkyl,
wherein aryl is defined as immediately above, and wherein the aryl may be mono and di-substituted, the substituents being each independently C1-6alkyl, halo, hydroxy, C1-6alkyl amino, C1-6alkoxy, C1-6alkylthio, and C1-6alkylcarbonyl;
AA4 is
an amino acid of formula AIV 
xe2x80x83wherein R4 selected from the group consisting of
(a) hydrogen, or
(b) substituted C1-6 alkyl, wherein the substituent is selected from
(1) hydrogen,
(2) hydroxy,
(3) halo,
(4) xe2x80x94Sxe2x80x94C1-4alkyl,
(5) xe2x80x94Oxe2x80x94C1-4alkyl
(6) xe2x80x94SH
(7) carboxy, 
(9) imidazolyl,
(10) guanidino, and
(11) amino;
n is an interger from 0-16 and
(AA)n is a peptide of 0-16 (ie n) amino acids in length, each amino acid being independent of formula 
xe2x80x83wherein X selected from the group consisting of
(a) hydrogen,
(b) substituted C1-6 alkyl, wherein the substituent is selected from
(1) hydrogen,
(2) hydroxy,
(3) halo,
(4) xe2x80x94Sxe2x80x94C1-4alkyl,
(5) xe2x80x94SH
(6) C1-6 alkylcarbonyl,
(7) carboxy, 0
(8) xe2x80x94CNH2,
(9) amino carbonyl amino,
(10) C1-4 alkylamino, wherein the alkyl moeity is substituted with hydrogen or hydroxy, and the amino is substituted with hydrogen or CBZ,
(11) guanidino,
(12) amino, and
(c) aryl C1-6 alkyl,
wherein the aryl group is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl,
(4) furyl,
(5) thienyl,
(6) thiazolyl,
(7) isothiazolyl,
(8) imidazolyl,
(9) benzimidazolyl,
(10) pyrazinyl,
(11) pyrimidyl,
(12) quinolyl,
(13) isoquinolyl,
(14) benzofuryl,
(15) benzothienyl,
(16) pyrazolyl,
(17) indolyl,
(18) purinyl,
(19) isoxazolyl, and
(20) oxazolyl,
and wherein the aryl may be mono and di-substituted, the substituents being each independently C1-6alkyl, halo, hydroxy, C1-6alkyl amino, C1-6alkoxy, C1-6alkylthio, and C1-6alkylcarbonyl;
R5 is
(a) substituted C1-12 alkyl, wherein the substituent is selected from
(1) hydrogen,
(2) hydroxy,
(3) halo, and
(4) C1-6alkylcarbonyl;
(b) aryl C1-6alkyl wherein the aryl group is selected from the group consisting of:
(1) phenyl,
(2) naphthyl,
(3) pyridyl,
(4) furyl,
(5) thienyl,
(6) thiazolyl,
(7) isothiazolyl,
(8) imidazolyl,
(9) benzimidazolyl,
(10) pyrazinyl,
(11) pyrimidyl,
(12) quinolyl,
(13) isoquinolyl,
(14) benzofuryl,
(15) benzothienyl,
(16) pyrazolyl,
(17) indolyl,
(18) purinyl,
(19) isoxazolyl, and
(20) oxazolyl,
and mono and di-substituted aryl as defined above in items (1) to (20) wherein the substitutents are independently C1-6alkyl, halo, hydroxy, C1-6alkyl amino, C1-6alkoxy, C1-6alkylthio, and C1-6alkylcarbonyl.
R6 is selected from the group consisting of:
(a) mono di or trisubstituted Aryl amino,
(b) mono di or trisubstituted Aryl oxy, and
(c) mono di or trisubstituted Aryl oxy,
wherein the aryl group is selected from the group consisting of: 
wherein the substituent is selected from the group consisting of
(1) H,
(2) OH,
(3) halo,
(4) C1-6alkyl,
(5) C1-6alkyloxy,
(6) CO2H,
(7) NO2 
(8) SO3H
(9) formyl,
(10) NH2,
(11) SH,
(12) C1-6alkylthio, 
(14) phenyl,
(15) phenylC1-6alkyl
(16) NO,
(17) C1-6alkylcarbonyl,
(18) phenylazo,
(19) C1-6sulfinyl,
(20) C1-6sulphonyl,
(21) phenyl sulfinyl,
(22) phenyl sulfonyl,
(23) phenyl carbonyl,
(24) phenyl oxy,
(25) phenyl thiol,
(26) C1-4alkylamino,
(27) diC1-4alkylamino, and
(28) CN,
and R8 is
C1-6alkyl or aryl C1-6alkyl wherein the aryl is selected form the group consisting of phenyl or naphthyl.
For purposes of this specification, (AA)n defines a peptide of 0,1,2,3,4,5,6,7,8,9,10,11,12,13, 14,15, or 16 amino acids in length. Similarly, for purposes of this specification, the amino acids AAI, AAII, AAIII, and AAIV may be each independently selected from the group consisting of the L- and D- forms of the amino acids including glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxy-lysine, histidine, arginine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, ornithine, xcex2-alanine, homoserine, homotyrosine, homophenylalanine and citrulline.
In one class of the second embodiment n is 0. Within this class is the subclass
wherein
AA1, is an amino acid of formula 
AA2 is an amino acid of formula 
AA3 is an amino acid of formula 
Within the subclass are the compounds wherein R2 and R3 are each independently selected from the group consisting of
(a) hydrogen,
(b) C1-6alkyl, wherein the substituent is selected from
(1) hydrogen,
(2) hydroxy,
(3) halo,
(4) xe2x80x94Sxe2x80x94C1-4alkyl
(5) xe2x80x94SH
(6) C1-6 alkylcarbonyl,
(7) carboxy, 
(9) C1-4alkylamino, and C1-4 alkyl amino wherein the alkyl moeity is substituted with an hydroxy, and
(10) guanidino,
(11) amino, and
(c) aryl C1-6alkyl, wherein aryl is phenyl, naphthyl, pyridyl, furyl, thienyl, thiazolyl, isothiazolyl, benzofuryl, benzothienyl, indolyl, isoxazolyl, and oxazolyl; and wherein the aryl may be mono and di-substituted, the substituents being each independently C1-6alkyl, halo, hydroxy, C1-6alkyl amino, C1-6alkoxy, C1-6alkylthio, and C1-6alkylcarbonyl.
R6 is selected from the group consisting of:
(a) mono or di substituted Aryl amino,
(b) mono or di substituted Aryl oxy, and
(c) mono or di substituted Aryl oxy,
wherein the aryl group is selected from the group consisting of: 
wherein the substituent is selected from the group consisting of
(1) H,
(2) OH,
(3) halo,
(4) C1-6alkyl,
(5) C1-6alkyloxy,
(6) CO2H,
(7) NO2,
(8) SO3H,
(9) formyl, and
(10) CN.
More particularly, illustrating the invention are the compounds wherein:
R5 is methyl;
R2 is C1-6alkyl; and
R3 is
(a) hydrogen,
(b) C1-6alkyl,
(d) benzyl,
(e) p-hydroxy-benzyl,
(f) N-carbobenzoxy-amino-(n-butyl),
(g) carbamylmethyl,
(h) carbamylethyl,
(i) indol-2-yl-methyl,
(j) substituted phenyl C1-6alkyl, wherein the substituent is hydrogen, hydroxy, carboxy, or C1-4alkyl,
(k) substituted indolyl C1-6alkyl, wherein the substituent is hydrogen, hydroxy, carboxy, or C1-4alkyl, or
(1) substituted imidazolyl C1-6alkyl wherein the substituent is hydrogen, hydroxy, carboxy, or C1-4alkyl.
In a third embodiment, the invention concerns a method of using a chromophore containing compound of formula I for determining the interleukin-1xcex2 converting enzyme activity of a sample, comprising:
(a) adding, in aqueous solution, in any order, (1) a compound of formula I, (2) interleukin-1xcex2 converting enzyme, and
(3) said sample; and
(b) measuring the interleukin-1xcex2 converting enzyme activity of the product of step (a) by photometric means.
The useful concentration of compound of Formula I in aqueous solution is 1 xcexcM to 10 mM. Typically, stock solutions are prepared in an organic solvent, such as DMSO, ethanol or isopropanol and diluted at least 20-fold in aqueous solution to achieve the desired concentration of substrate in the reaction mixture. The enzyme tolerates concentrations of some organic solvents (ethanol, isopropanol, DMSO) up to 20% (vol/vol) with no significant loss of enzyme activity. The choice of solvent is dictated entirely by the concentration desired in the assay, and the solubility of the substrate. Alternatively, the substrate stock solution could be prepared in buffer at a dilute concentration, and comprise a large percentage of the final reaction mixture.
Similarly, it is preferred that the aqueous solution comprises a buffer.
The pH optimum for ICE is between 6.5 and 7.5. Consequently, suitable buffer will have a pKa between 6.5 and 7.5, such as HEPES, which we use in our studies. In general, any nonreactive buffer at a concentration that will maintain the pH of the reaction between 6 and 9 will work.
Other components may be added to the reaction that stabilize the enzyme or increase the rate of the reaction. Examples are sucrose (10%), CHAPS (0.1%), DTT (1-100 mM), BSA (0.1-10 mg/ml) all of which have been demonstrated to stabilize the enzyme. Others components which may be included are glycerol, EDTA, and a variety of standard protease inhibitors.
The concentration of ICE is highly variable and may range from 1pM to 1 xcexcM, depending entirely on the purpose of a particular experiment, and the kinetic parameters for the chosen substrate. The volume added to a particular reaction may be very small or comprise the entire volume of the reaction less the volume of substrate required to achieve the desired concentration.
Enzyme for use in the method may be obtained from any cell capable of secreting IL1B such as those listed in the Background of the Invention. Any state of purity of ICE is acceptable (including crude cell lysates), as long as the preparation is free of contaminating proteases that will compete with ICE for cleavage of the substrate. Even in this case it is possible to use this assay if inhibitors of the contaminating proteases are included in the reaction.
The sample will typically comprise either, a putative ICE inhibitor, in a concentration of 1pM to 1M or any other modulator of ICE activity.
This assay is typically run between 25 and 37 degrees. The use of higher temperatures will depend upon the stability of the enzyme and running the assay at low temperatures will probably be dictated by practical considerations.
As appreciated by those of skill in the art, addition step (a) results in the cleavage of compound of formula I between the aspartic acid specifically described, and the adjacent group, R6. The liberation of the chromophoric group, R6 may be monitored by spectrophotometric or fluorometric procedures.
The method of detection will depend upon the chromophore released upon hydrolyis of the Asp-X bond. Fluorometric leaving groups (e.g. AMC) require spectrofluorometer such as the Gilford Fluoro IV. The emission and excitation wavelengths will be selected based on the emission and excitation spectra of the substrate and product chromophore. In the case of Ac-Tyr-Val-Ala-Asp-AMC, the excitation wavelength is 380 nm and the emission wavelength is 460nm.
Substrates with spectrophotometric leaving groups (eg. pNA) will require a spectrophotometer such as a CARY 210 spectrophotometer. In this case the reaction will be monitored at a wavelength whose selection will be based on the absorbance spectra of the substrate and product chromophore. In the case of Ac-Tyr-Val-Ala-Asp-PNA, the wavelength selected is 410 nm, although this can vary appreciably with only a minor compromise in the sensitivity of the assay.
In general, the fluorometric assays will be 10-fold more sensitive than spectrophotometric assays, consequently, the fluorometric assay is preferred if enzyme is precious. However, in the event that large quantities of active recombinant protein become available, the spectrophotometric assays are preferred. This assay is amenable to continuous or discontinuous sampling of the reaction. The assay is also amenable to 96-well plate format for running multiple assays simultaneously.
For example, with the fluorometric leaving group (e.g. AMC), the activity of the sample is proportional to the rate of fluorescence change, and be calculated as:                               Velocity of                                              ICE          ⁢                      xe2x80x83                    ⁢                      Catalyzed                                                        reaction                      =                              ⅆ                      fluorescense                                    ⅆ          t                    ⁢              (                              1            ⁢            μ            ⁢                          xe2x80x83                        ⁢            M            ⁢                          xe2x80x83                        ⁢            AMC                    fluorescence                )              =                  ⅆ        AMC                    ⅆ        t            
As appreciated by those of skill in the art, the use described above may be quite useful for determining Michaelis-Menton kinetic parameters or other characterization information concerning the Enzyme (eg when the sample contains no putatuve inhibitor) or screening for putative ICE inhibitors or assaying purification fractions.
In a fourth embodiment, the invention concerns a method of using a chromophore containing compound of formula II for determining the interleukin-1xcex2 converting enzyme activity of a sample, comprising:
(a) adding, in aqueous solution, in any order, (1) a compound of formula II, (2) interleukin-1xcex2 converting enzyme, (3) a sample, and (4) an aminopeptidase; and
(b) measuring the interleukin-1xcex2 converting enzyme activity of the product of step (a) by spectrophotometric or fluorometric analysis.
With the exception of the compound of Formula II and selection and concentration of components is the same as that stated for the third embodiment. With regard to the peptidase, any peptidase capable of cleaning the bond between AA4 and R6 will prove satisfactory.
Applicants have found a leucine aminopeptidase (LAPM) isolated from kidney microsomes (SIGMA CHEMICAL Co. No. L-0632) to be quite satisfactory.
It is not inherently essential that the sample and the LAPM be added at the same time. It is essential that the rate of hydrolysis by LAPM is not the rate limiting step of the overall reaction. We have found it useful to monitor the reaction continuously. The concentration of LAPM typically used in the assay is 1 xcexcL, however, the amount used may vary widely (e.g. 0.01 units to 100 units per xcexc1 depending on the substitute and/or sample). [1 unit will hydrolyze 1.0 xcexcmole of L-leucine-p-nitroaniline to leucine and p-nitroaniline per minute at pH 7.2 at 37xc2x0 C.]
A chromophore can be coupled to a suitably protected aspartic acid derivative as shown in Scheme I. p-Nitrobenzoic acid is treated with DPPA in the presence of triethyl amine to effect a Curtius rearrangement. The resulting p-nitrophenylisocyanate reacts with N-trimethylsilylethyloxycarbonyl aspartic acid b-t-butyl ester to form the corresponding p-nitroanilide. The urethane is removed with tetrabutyl ammonium fluoride and the resulting amine coupled to (N-acetyl-tyrosinyl)-valinyl-aline using DCC and HOBt. The t-butyl ester is then removed with trifluoroacetic acid to provide the desired chromogenic peptide. 
A chromophore can also be coupled to a suitably protected aspartic acid derivative as shown in Scheme II. 6-Aminoquinoline reacts with FMOC-aspartic acid B-t-butyl ester to form the corresponding amide in the presence of EDC and DMAP. The FMOC group is removed with diethyl amine and the resulting amine coupled to (N-acetyl-tyrosinyl)-valinyl-alanine using DCC and HOBt. The t-butyl ester is then removed with trifluoroacetic acid to provide the desired chromogenic peptide. 
A chromophore can also be coupled to a suitably protected aspartic acid derivative as shown in Scheme III. 7-Amino-4-methylquinoline reacts with Alloc-aspartic acid B-t-butyl ester to form the corresponding amide in the presence of EDC. The Alloc group is removed with tetrakis triphenylphosphine palladium and dimedone and the resulting amines coupled to (N-acetyl-tyrosinyl)-valinyl-aline using DCC and HOBt. The t-butyl ester is then removed with trifluoroacetic acid to provide the desired chromogenic peptide. 
A chromogenic peptide substrate for IL-lB converting enzyme can also be prepared as shown in Scheme IV. (N-Acetyl-tyrosinyl)-valinyl-alaninyl-aspartic acid B-t-butyl ester can be coupled to commercially available glycine 7-amino-4-methylcoumarin amide using DCC and HOBt. The t-butyl ester is then removed with trifluoroacetic acid to provide the desired chromogenic peptide.
The following Examples are intended to illustrate the invention, and as such are not intended to limit the invention as set forth in the claims appended, thereto. The compounds of Formula I described in Examples 1A, 1B, 2, 6, 7 and 8 correspond to the peptide sequence which is SEQ. ID NO: 2:.
The compounds of Formula I described in Examples 3 and 5 correspond to the peptide sequence which is SEQ. ID NO: 3:.